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Recombinant plasmid and method for expressing hepatitis B viral antigens and virions in vivo

a plasmid and hepatitis b technology, applied in the field of recombinant plasmids and methods for expressing hepatitis b viral antigens and virions in vivo, can solve the problems of different mechanisms of liver inflammation derived from such hbv transgenic mice, cell apotosis, and animal models are not ideal for studying hbv tolerance and clearan

Active Publication Date: 2008-11-18
NAT TAIWAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is generally believed that HBV is not directly cytopathic to the hepatocytes, but HBV mediates the immune response of the infected hepatocytes and triggers the infected cells to express viral antigen, which resulting in host immune system attacking its own infected hepatocytes and eventually leading to cell apotosis.
However, it has been found that the mechanism of liver inflammation derived from such HBV transgenic mice is different from scenario of human hepatitis.
Furthermore, such HBV transgenic mice are HBV antigen tolerant, thus do not exhibit normal immune response against HBV.
Hence, it is concluded that such animal models are not ideal in study of HBV tolerance and clearance.
However, there are disadvantages to this animal model, that is, short-term acute liver inflammatory response and clearance of immune system effect 14 days after transfection.
These constraints limit application of such animal model in studies such as long term monitoring of immune response after HBV infection and drug evaluation in chronic hepatitis.

Method used

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  • Recombinant plasmid and method for expressing hepatitis B viral antigens and virions in vivo
  • Recombinant plasmid and method for expressing hepatitis B viral antigens and virions in vivo
  • Recombinant plasmid and method for expressing hepatitis B viral antigens and virions in vivo

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Vector

[0031]The HBV 1.2-full-length DNA is isolated from the plasmid pHBV-48, containing a greater-than-genome-length HBV fragment (subtype adw, gene type A) cloned in pGEM-3Z vector. The replication-competent HBV 1.2-full-length gemone replaced the GFP fragment in AAV-GFP vector by ligation of the BamHI / EcoRI (1.8 kb) and EcoRI / BglII (2.0 kb) fragments of pHBV-48 to the BglII site of AAV-GFP vector. The resulting pAAV / HBV (1400) contains the HBV fragment spanning from nucleotides 1400-3182 / 1-1987 flanked by inverted terminal repeats (ITRs) of AAV.

example 2

Transfection Procedure and Determination

[0032]In vivo experiment of the vector transfection, 6 to 8 weeks old male C57BL / 6 mice with H-2b haplotype are used as experimental group, whereas 6 to 8 weeks old male BALB / c with H-2d haplotype are selected as control group. Ten micrograms of the abovementioned HBV plasmid pAAV / HBV (1400) were injected into the tail vein of mice in a volume of phosphate-buffered saline (PBS) equivalent to 8% of the mouse body weight under hydrodynamic condition that is well known for those skilled in the art. The total volume is delivered within 5 seconds.

[0033]After injection, the mice are regularly bled to follow serum concentration of HBV surface antigen (HBsAg), anti-HBV core protein antibodies (anti-HBc) and anti-HBs antibodies (anti-HBs) at the indicated time points, using the AXSYM system kit (Abbott, GmbH Diagnostica). The livers of mice are preserved in optimal cutting temperature (OCT) for immunohistochemical analysis. Expression of HBcAg and HBsA...

example 3

[0036]The HBV expression cassette is excised by SmaI digestion (located inside of the two ITRs) from the pAAV / HBV (1400) and subcloned into SmaI site of pGEM4Z to result in pGEM4Z / HBV (1400). Then the obtained recombinant plasmid pGEM4Z / HBV (1400) is injected into the tail vein of C57BL / 6 mice according to the method described in example 2.

[0037]The results show that the injection of pGEM4Z / HBV (1400) into C57BL / 6 mice produced only transient surface antigenemia (shown in FIG. 1b) and all of them developed anti-HBs within 4 weeks (Table 1), suggesting that persistence of HBV in C57BL / 6 mice is associated with of injection of pAVV / HBV (1400) plasmid.

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Abstract

Disclosed is a recombinant plasmid for expressing hepatitis B viral antigens in vivo, comprising an adeno-associated virus (AVV) vector and a replication-competent hepatitis B virus genome fragment. Mice hydrodynamically injected with the recombinant plasmid of the present invention show persistent expression of hepatitis B viral antigens for more than 6 months in the hepatocytes, thus a immuno-competent mouse model for persistent expression of hepatitis B antigens and also for human chronic hepatitis B virus infection is established, which can be applied in evaluation and elucidation of mechanism of chronic hepatitis and anti-viral drug discovery research.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a method for expressing hepatitis B viral antigens and virions in vivo, especially relates to a recombinant plasmid for expressing all of the hepatitis B viral antigens and a method to construct an immunocompetent mouse model for persistently expressing hepatitis B vial antigens by using the recombinant plasmid.[0003]2. The Prior Arts[0004]It is estimated that 350 million people are chronic carriers of hepatitis B virus worldwide, and there are approximately 1 million deaths each year caused by hepatitis B related liver diseases. Evidently, hepatitis B virus has tremendous impact on human health, therefore research for best treatment and new drug discovery enthusiastically become an important healthcare issue.[0005]Hepatitis B virus (HBV) is a partially double-stranded DNA virus that infects human hepatocytes. Upon infection of host individual, the hepatitis B virus will first form a cov...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/09A01N63/00A61K39/12C12P21/04
CPCA61K39/292C07K14/005A61K2039/53C12N2730/10122C12N2730/10134C12N2750/14143A61K39/12
Inventor CHEN, PEI-JERHUANG, LI-RUNGWU, HUI-LINCHEN, DING-SHINN
Owner NAT TAIWAN UNIV
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